The continuous generation of B lymphocytes is required for a functional immune system. In aged humans, the adaptive immune response is significantly compromised. The consequence of this decline for human health is poor efficacy of vaccination and increased vulnerability to infection. Decreased production of new B cells contributes to the age- associated decline of the immune system. In aged mice, inefficient transition from pro- to pre-B cells account of a significant component of the decline. Pro-B cells undergo V(D)J recombination to generate a fully assembled immunoglobulin heavy chain gene. This process is dependent upon gene expression of V(D)J recombinase activating genes RAG1 and RAG2. We propose that diminished RAG gene expression occurs in pro- B cells in aging mice, the result of an age-associated decline in the hematopoietic microenvironment that regulates RAG gene expression through extrinsic factors, and that this decline is an underlying mechanism in decreased B cell generation during aging. We will test this hypothesis by addressing two Specific Aims: One: determine whether there is a reduction in RAG2 transcription specifically in the pro-B ells of aged mice. Two: test whether environmental changes or cell-intrinsic during aging are primarily responsible for the decline in RAG2 expression and B cell development. To accomplish these aims, we will utilize a strain of transgenic mice in which the Green Fluorescent Protein (GFP) reporter gene is under the transcriptional control of the RAG2 genomic locus. This model system allows for the first time flow cytometric detection of RAG2 gene expression at the single cell level in conjunction with surface markers that define stages of B cell development. Our approach has two major benefits; the direct measurement of transcription activity in live cells, and the use of single- cell analyses to determine the fraction of cells at various stages of B cell development in which REAG2 gene expression occurs and is compromised in aging. This work uses new techniques to study the effect of aging on the immune system and intends, as a long term goal, to identify mechanisms responsible for diminished B cell production in aged mice and humans.